Potassium permeability of Rickettsia prowazekii.

The potassium permeability of Rickettsia prowazekii was characterized by chemical measurement of the intracellular sodium and potassium pools and isotopic flux measurements with 86Rb+ as a tracer. R. prowazekii, in contrast to Escherichia coli, did not maintain a high potassium-to-sodium ratio in their cytoplasm except when the potassium-to-sodium ratio in the extracellular medium was high or when the extracellular concentrations of both cations were low (ca. 1 mM). Both influx and efflux assays with 86Rb+ demonstrated that the rickettsial membrane had limited permeability to potassium and that incorporation of valinomycin into these cells increased these fluxes at least 10-fold. The transport of potassium showed specificity and dependence on rickettsial metabolism. The increased flux of potassium which results from the incorporation of valinomycin into the rickettsial membrane was detrimental to both lysine transport and lysis of erythrocytes by the rickettsiae.     

Biochemical and immunochemical study of antigen preparations of Rickettsia prowazekii

The biochemical and immunochemical study of the qualitative composition of R. prowazekii antigenic preparations, isolated and purified by different methods, indicates that these preparations contain high-molecular polypeptide 3 (133600 D), morphologically linked with the cell membrane of R. prowazekii. The method of adsorption chromatography on calcium phosphate permits obtaining specific rickettsial antigens with a greater degree of purification from ballast admixtures than the methods of acid precipitation and gel filtration on Sephadex G-200.     

Transport of AMP by Rickettsia prowazekii.

Rickettsia prowazekii possesses an exchange transport system for AMP. Chromatographic analysis of the rickettsiae demonstrated that transported AMP appeared intracellularly as AMP, ADP, and ATP, and no hydrolytic products appeared in either the intracellular or extracellular compartments. The phosphorylation of AMP to ADP and ATP was prevented by pretreatment of the cells with 1 mM N-ethylmaleimide without inhibiting the transport of AMP. Although no efflux was demonstrable in the absence of nucleotide in the medium, the intracellular adenine nucleotide pool could be exchanged with external unlabeled adenine nucleotides. Both ADP and ATP were as effective as AMP at inhibiting the uptake of [3H]AMP. Although this transport system was inhibited by low temperature (0 degrees C) and partially inhibited by the protonophore carbonyl cyanide-m-chlorophenyl hydrazone (1 mM), it was relatively insensitive to KCN (1 mM). The uptake of AMP at 34 degrees C had an apparent Kt for influx of 0.4 mM and a Vmax of 354 pmol min-1 per mg. At 0 degrees C there was a very rapid and unsaturable association of AMP with these organisms. Correction of the uptake data at 34 degrees C for the 0 degrees C component lowered the apparent Kt to 0.15 mM. Both magnesium and phosphate ions are required for optimal transport activity. Chemical measurements of the total intracellular nucleotide pools demonstrated that this system was not a net adenine nucleotide transport system, but that uptake of AMP was the result of an exchange with internal adenine nucleotides.     

Permeability of Rickettsia prowazekii to NAD.

Rickettsia prowazekii accumulated radioactivity from [adenine-2,8-3H]NAD but not from [nicotinamide-4-3H]NAD, which demonstrated that NAD was not taken up intact. Extracellular NAD was hydrolyzed by rickettsiae with the products of hydrolysis, nicotinamide mononucleotide and AMP, appearing in the incubation medium in a time- and temperature-dependent manner. The particulate (membrane) fraction contained 90% of this NAD pyrophosphatase activity. Rickettsiae which had accumulated radiolabel after incubation with [adenine-2,8-3H]NAD were extracted, and the intracellular composition was analyzed by chromatography. The cells contained labeled AMP, ADP, ATP, and NAD. The NAD-derived intracellular AMP was transported via a pathway distinct from and in addition to the previously described AMP translocase. Exogenous AMP (1 mM) inhibited uptake of radioactivity from [adenine-2,8-3H]NAD and hydrolysis of extracellular NAD. AMP increased the percentage of intracellular radiolabel present as NAD. Nicotinamide mononucleotide was not taken up by the rickettsiae, did not inhibit hydrolysis of extracellular NAD, and was not a good inhibitor of the uptake of radiolabel from [adenine-2,8-3H]NAD. Neither AMP nor ATP (both of which are transported) could support the synthesis of intracellular NAD. The presence of intracellular [adenine-2,8-3H]NAD within an organism in which intact NAD could not be transported suggested the resynthesis from AMP of [adenine-2,8-3H]NAD at the locus of NAD hydrolysis and translocation.     

Analysis of the peptidoglycan of Rickettsia prowazekii.

In the present study, peptidoglycan from Rickettsia prowazekii, an obligate intracellular bacterium, was purified. The rickettsial peptidoglycan is like that of gram-negative bacteria; that is, it is sodium dodecyl sulfate insoluble, lysozyme sensitive, and composed of glutamic acid, alanine, and diaminopimelic acid in a molar ratio of 1.0:2.3:1.0. The small amount of lysine found in the peptidoglycan preparation suggests that a peptidoglycan-linked lipoprotein(s) may be present in the rickettsiae. D-Cycloserine, a D-alanine analog which inhibits the biosynthesis of bacterial cell walls, prevented rickettsial growth in mouse L929 cells at a high concentration and altered the morphology of the rickettsiae at a low concentration. These effects were prevented by the addition of D-alanine. This suggests that R. prowazekii contains D-alanine in the peptidoglycan and has D-Ala-D-Ala ligase and alanine racemase activities.     

Instability of Rickettsia prowazekii RNA polymerase-promoter complexes.

The Rickettsia prowazekii sigma factor was overexpressed, purified, and used to reconstitute RNA polymerase holoenzyme species. R. prowazekii RNA polymerase-promoter complexes were unstable and remained dissociable and heparin sensitive under conditions in which the corresponding Escherichia coli complexes were not. The R. prowazekii core played the major role in determining heparin sensitivity.     

Isolation and characterization of the Rickettsia prowazekii recA gene.

The recA gene has been isolated from Rickettsia prowazekii, an obligate intracellular bacterium. Comparison of the amino acid sequence of R. prowazekii RecA with that of Escherichia coli RecA revealed that 62% of the residues were identical. The highest identity was found with RecA of Legionella pneumophila, in which 69% of the residues were identical. Amino acid residues of E. coli RecA associated with functional activities are conserved in rickettsial RecA, and the R. prowazekii recA gene complements E. coli recA mutants for UV light and methyl methanesulfonate sensitivities as well as recombinational deficiencies. The characterized region upstream of rickettsial recA did not contain a sequence homologous to an E. coli LexA binding site (SOS box), suggesting differences in the regulation of the R. prowazekii recA gene.     

S-adenosylmethionine transport in Rickettsia prowazekii

Rickettsia prowazekii, the causative agent of epidemic typhus, is an obligate, intracellular, parasitic bacterium that grows within the cytoplasm of eucaryotic host cells. Rickettsiae exploit this intracellular environment by using transport systems for the compounds available in the host cell's cytoplasm. Analysis of the R. prowazekii Madrid E genome sequence revealed the presence of a mutation in the rickettsial metK gene, the gene encoding the enzyme responsible for the synthesis of S-adenosylmethionine (AdoMet). Since AdoMet is required for rickettsial processes, the apparent inability of this strain to synthesize AdoMet suggested the presence of a rickettsial AdoMet transporter. We have confirmed the presence of an AdoMet transporter in the rickettsiae which, to our knowledge, is the first bacterial AdoMet transporter identified. The influx of AdoMet into rickettsiae was a saturable process with a K(T) of 2.3 micro M. Transport was inhibited by S-adenosylethionine and S-adenosylhomocysteine but not by sinfungin or methionine. Transport was also inhibited by 2,4-dinitrophenol, suggesting an energy-linked transport mechanism, and by N-ethylmaleimide. AdoMet transporters with similar properties were also identified in the Breinl strain of R. prowazekii and in Rickettsia typhi. By screening Escherichia coli clone banks for AdoMet transport, the R. prowazekii gene coding for a transporter, RP076 (sam), was identified. AdoMet transport in E. coli containing the R. prowazekii sam gene exhibited kinetics similar to that seen in rickettsiae. The existence of a rickettsial transporter for AdoMet raises intriguing questions concerning the evolutionary relationship between the synthesis and transport of this essential metabolite.     

Unusual organization of the rRNA genes in Rickettsia prowazekii.

We describe here the organization of the rRNA genes in Rickettsia prowazekii. In this organism, the 23S and the 5S rRNA genes are tightly linked to each other, whereas the 16S rRNA gene is separated from this cluster. The 23S-5S unit is preceded by the methionyl-tRNAfMet formyltransferase gene.     

Action of tetracycline and rifampicin on Rickettsia prowazekii and Rickettsia sibirica

The effect of tetracycline and rifampicin on R. prowazekii, strain Breinl and R. sibirica, strain X1 was studied in the experiments with chick embryos exposed to the antibiotic mixture with the infection material. It was shown that tetracycline in doses of 0.1 and 1 mg/embryo had the rickettsiostatic and rickettsiocidic effects respectively on R. sibirica. Rifampicin had only the rickettsiostatic effect in a dose of 0.1 mg/embryo and no rickettsiocidic effect even in a dose of 2 mg/embryo. Higher doses were toxic for 100 per cent of the embryos. The rickettsiostatic and rickettsiocidic effects of tetracycline on R. prowazekii were evident in doses of 0.05 and 0.1 mg/embryo, respectively. Rifampicin in a dose of 0.05 mg/embryo had both the rickettsiostatic and the rickettsiocidic effects on R. prowazekii. Therefore, rifampicin was more active with respect to R. prowazekii and tetracycline was more active with respect to R. sibirica. In addition, R. sibirica was more resistant to both tetracycline and rifampicin as compared to R. prowazekii.     

The library of Rickettsia prowazekii genes

The DNA of Rickettsia provazekii strain E was cleaved by PstI restriction endonuclease under the conditions of partial restriction. The fragments were inserted into the PstI site of pBR325 and cloned in this plasmid. E. coli strain HB101 was used as a recipient for cloning. 880 clones sensitive to ampicillin and resistant to tetracycline were selected from 5120 transformants. The cloning of rickettsial DNA has been confirmed by the blot hybridization technique. Analysis of individual and net probes of the hybrid DNA by gel electrophoresis makes it possible to conclude that 90% of the selected clones harbour hybrid plasmids, the size of the cloned fragments rangers from 0.9 to 10.4 Kb, the obtained library of clones contains 70% of the whole genome of Rickettsia provazekii.     

Mariner-based transposon mutagenesis of Rickettsia prowazekii

Rickettsia prowazekii, the causative agent of epidemic typhus, is an obligate intracellular bacterium that grows directly within the cytoplasm of its host cell, unbounded by a vacuolar membrane. The obligate intracytoplasmic nature of rickettsial growth places severe restrictions on the genetic analysis of this distinctive human pathogen. In order to expand the repertoire of genetic tools available for the study of this pathogen, we have employed the versatile mariner-based, Himar1 transposon system to generate insertional mutants of R. prowazekii. A transposon containing the R. prowazekii arr-2 rifampin resistance gene and a gene coding for a green fluorescent protein (GFP(UV)) was constructed and placed on a plasmid expressing the Himar1 transposase. Electroporation of this plasmid into R. prowazekii resulted in numerous transpositions into the rickettsial genome. Transposon insertion sites were identified by rescue cloning, followed by DNA sequencing. Random transpositions integrating at TA sites in both gene coding and intergenic regions were identified. Individual rickettsial clones were isolated by the limiting-dilution technique. Using both fixed and live-cell techniques, R. prowazekii transformants expressing GFP(UV) were easily visible by fluorescence microscopy. Thus, a mariner-based system provides an additional mechanism for generating rickettsial mutants that can be screened using GFP(UV) fluorescence.     

Study of the immunobiological properties of a soluble antigen of Rickettsia prowazekii in an experiment

The results obtained in the study of the immunobiological properties of R. prowazekii soluble antigen purified without the use of ether are presented. The effectiveness of this antigen has been shown to be not inferior to that of preparations obtained by ether purification. It is expedient to introduce the antigen in the adsorbed form in two injections at an interval of 10--14 days, as immunization in two injections produces phase changes in the immune responsiveness of animals.